Glycogen synthase kinase 3ß participates in late stages of Dengue virus-2 infection

BACKGROUND Viruses can modulate intracellular signalling pathways to complete their infectious cycle. Among these, the PI3K/Akt pathway allows prolonged survival of infected cells that favours viral replication. GSK3β, a protein kinase downstream of PI3K/Akt, gets inactivated upon activation of the PI3K/Akt pathway, and its association with viral infections has been recently established. In this study, the role of GSK3β during Dengue virus-2 (DENV-2) infection was investigated. METHODS GSK3β participation in the DENV-2 replication process was evaluated with pharmacological and genetic inhibition during early [0-12 h post-infection (hpi)], late (12-24 hpi), and 24 hpi in Huh7 and Vero cells. We assessed the viral and cellular processes by calculating the viral titre in the supernatants, In-Cell Western, western blotting and fluorescence microscopy. RESULTS Phosphorylation of GSK3β-Ser9 was observed at the early stages of infection; neither did treatment with small molecule inhibitors nor pre-treatment prior to viral infection of GSK3β reduce viral titres of the supernatant at these time points. However, a decrease in viral titres was observed in cells infected and treated with the inhibitors much later during viral infection. Consistently, the infected cells at this stage displayed plasma membrane damage. Nonetheless, these effects were not elicited with the use of genetic inhibitors of GSK3β. CONCLUSIONS The results suggest that GSK3β participates at the late stages of the DENV replication cycle, where viral activation may promote apoptosis and release of viral particles.

Nuclei ultrastructural changes of C6/36 cells infected with virus dengue type 2 / Cambios ultraestructurales en núcleos de células C6/36 infectadas con virus dengue de tipo 2

ABSTRACT Introduction: Dengue virus replication has been considered mainly cytoplasmic, however, studies indicate that some flaviviruses may use the intranuclear pathway as part of the machinery that the virus uses to increase infection capacity in the host cell. This paper describes alterations at nuclear level in the cell infected with dengue, which are likely involved in the virus replication processes. Objective: This paper addresses the ultrastructural observations of C6/36 cells of the Aedes albopictus mosquito infected with dengue virus type 2. Materials and methods: C6/36 cells were infected in culture medium with the serum of a patient positively diagnosed for dengue 2. Subsequently, the cells were incubated for 10 days and the cytopathic effect was assessed. The cells were processed for immunofluorescence assays and transmission electron microscopy. Results: The immunofluorescence assays confirmed the presence of viral protein E associated with cellular syncytia in the culture. In the ultrastructural study, the infected cells showed vesicular-tubular structures and dilated cisterns of the endoplasmic reticulum at the cytoplasmic level. Viral particles were found exclusively in cytoplasm localized within the vacuoles. Nuclei of cellular syncytia showed membrane structures arranged in a circular shape and, in some cases, these syncytia displayed lysis; in no case viral particles were observed at the nuclear level. Conclusions: The ultrastructural alterations of nuclei in cells infected with the dengue virus using electron microscopy techniques had not been reported before, as far as we know. It is likely that such modifications are associated with replicative processes at an intranuclear level as an alternate replication mechanism.

RESUMEN Introducción. La replicación del virus del dengue se ha considerado principalmente citoplásmica; sin embargo, en diversos estudios se ha informado que algunos flavivirus pueden utilizar factores intranucleares como parte de la maquinaria que utilizan para aumentar la capacidad de infección en la célula huésped. En este trabajo se describen las alteraciones a nivel nuclear en células infectadas con dengue, probablemente involucradas en procesos de replicación viral. Objetivo. Presentar las observaciones ultraestructurales de células C6/36 de Aedes albopictus infectadas con el virus del dengue de tipo 2. Materiales y métodos. Se infectaron células C6/36 con suero de un paciente con diagnóstico de dengue 2; posteriormente, se mantuvieron en medio de cultivo durante 10 días y se evaluó el efecto citopático. Las células se procesaron para los ensayos de inmunofluorescencia y microscopía electrónica de transmisión, con el fin de hacer el estudio ultraestructural. Resultados. Los ensayos de inmunofluorescencia confirmaron la presencia de la proteína E viral asociada con sincitios celulares en el cultivo. En el estudio ultraestructural, las células infectadas tenían estructuras vesiculares y tubulares, y cisternas dilatadas del retículo endoplásmico en el citoplasma. Las partículas virales se encontraron exclusivamente en vacuolas localizadas en el citoplasma. Los núcleos de los sincitios celulares contenían estructuras de membrana dispuestas en forma circular y, en algunos casos, dichos sincitios presentaban lisis. En ningún caso se observaron partículas virales en el núcleo. Conclusiones. No se habían reportado alteraciones ultraestructurales en los núcleos de células infectadas con el virus del dengue detectadas mediante técnicas de microscopia electrónica. Es probable que tales modificaciones estén asociadas con procesos intranucleares de replicación como un mecanismo alternativo.

Selección de variantes de virus dengue sensibles a la heparina en células BHK-21 / Selection of heparin-sensitive dengue virus variants in BHK-21 cells

Estudios previos han demostrado que la adaptación de diversos virus a crecer en líneas celulares de vertebrados, conduce a la selección de variantes virales que unen al heparán sulfato (HS) con alta afinidad. En el presente trabajo se determinó la susceptibilidad de cepas del virus dengue (DENV) a la heparina hipersulfatada un análogo al HS, después de pases seriados en células BHK-21. A aislados de campo de los cuatro serotipos de DENV, se les realizaron ocho pases seriados en células BHK-21. La adaptación de los DENV al cultivo celular seleccionó variantes virales con una aumentada capacidad replicativa en células BHK-21 y una incrementada susceptibilidad a la heparina, en relación a las respectivas cepas no adaptadas, obteniéndose una inhibición de la infectividad más significativa en DENV-2 y DENV-4. Las cepas de DENV adaptadas presentaron cambios en la secuencia de aminoácidos de la proteína de envoltura (E), en particular una substitución K204R para DENV-1, N67K para DENV-2, K308R y V452A para DENV-3 y E327G para DENV-4. Estas sustituciones implicaron ganancia de residuos básicos que incrementaron la carga neta positiva de la proteína. Los resultados sugieren, que la adaptación de cepas de DENV a células BHK-21 selecciona variantes virales sensibles a la heparina y que la efectividad de este compuesto varía dependiendo de la cepa viral. Además sugieren que el HS puede jugar un papel importante en la infectividad de las cepas de DENV adaptadas al cultivo celular, a diferencia de los aislados de DENV no adaptados.

Several studies have shown that adaptation of various viruses to grow in certain cell lines of vertebrates, leads to the selection of virus variants that bind heparan sulfate (HS) with high affinity. In this study we investigated the susceptibility of strains of dengue virus (DENV) to oversulfated heparin an analogue of HS after passages in BHK-21 cells. Field isolates of the four serotypes of DENV with a limited number of passes in mosquito cells C6/36HT were serially passaged eight times in BHK-21 cells. The adaptation of the DENV to the cell culture selected viral variants with an increased replicative capacity in BHK-21 cells and an increased susceptibility to heparin compared with the original not adapted strains, with a more significant inhibition of the infectivity in DENV-2 and DENV-4.The E protein of the adapted strains showed changes in the amino acid sequence, particularly at the position K204R to DENV-1, N67K to DENV-2, K308R and V452A for DENV-3 and E327G to DENV-4. These substitutions implicated a gain of basic residues that increased the net positive charge of the protein. These results suggest that adaptation of DENV strains to BHK-21 cells implies changes in the envelope protein, changes associated to the protein reactivity with heparin, the inhibitory effectiveness of this compound varying depending on the viral strain. In addition, these results suggest that the HS can play an important role in the infectivity of the DENV strains adapted to vertebrate cell culture, but not in the infectivity of non-adapted DENV isolates.

Detecção da bactéria Wolbachia em insetos através da técnica LAMP (amplificação isotérmica mediada por loop) / Detection of Wolbachia bacteria in insects through the LAMP technique (isothermal amplification Loop-mediated)

Wolbachia pipientis é uma bactéria intracelular que infecta cerca de 40% dos artrópodes e alguns nematódeos, causando alterações reprodutivas em seus hospedeiros. Recentemente, duas cepas de Wolbachia (wMelPop e wMel)foram inseridas individualmente em Aedes aegypti (não infectado naturalmente)e os mosquitos adultos contendo a Wolbachia foram menos suscetíveis a diferentes arboviroses (Dengue e Chikungunya) bem como ao Plasmodium sp. Atualmente, mosquitos A. aegypti contendo Wolbachia (wMel) estão sendo liberados a campo em diversos países, pelo Programa Eliminate Dengue, como possível agente de controle biológico de Dengue. Durante o processo de invasão de mosquitos contendo Wolbachia no campo, há a necessidade de realização de coletas e screening semanal de grandes quantidades de mosquitos para detecção de Wolbachia, via PCR quantitativo, técnica ainda bastante onerosa. Diante disto, é imprescindível o desenvolvimento de um método rápido, específico e de baixo custo para detecção de mosquitos infectados com a bactéria, para levantamento e monitoramento da disseminação da Wolbachia em campo. No presente trabalho, desenvolvemos com sucesso um método rápido de detecção através do LAMP (Amplificação isotérmica mediada por loop) que utiliza 3 pares de primers que se ligam em 8 regiões distintas do DNA alvo, o que torna a reação bastante específica. Em nosso caso, desenhamos iniciadores baseando-se na sequência alvo do gene 16S rRNA da bactéria. Para padronização, foram utilizados mosquitos da colônia do Insetário do Laboratório de Malária do CPqRR, infectados e não infectados por Wolbachia, além de insetos de campo (mosquitos e insetos de diferentes ordens) doados por colaboradores. O tempo de incubação estabelecido para a amplificação foi de 90 minutos à 63oC, sendo possível a realização da reação tanto em termociclador, como em banho seco...

Para análise da sensibilidade, foi feita a diluição seriada de plasmídeo contendo a sequência alvo de 109 à 100 cópias, sendo possível a amplificação utilizando apenas 1 cópia do DNA. Para verificar a especificidade, foram utilizadas amostras de diferentes espécies de bactérias e em nenhuma delas ocorreu a amplificação, confirmando que o LAMP é bastante específico. Uma maneira de reduzir os custos foi através da redução da concentração da enzima Bst DNA polimerase por reação e, com apenas 1 unidade, a amplificação ocorreu de maneira eficiente. Para visualização dos resultados, foram utilizados dois corantes, o azul de hidroxinaftol (HNB) e SYBR Green I. Em ambos foi possível diferenciar as amostras positivas das negativas sem a necessidade de géis de agarose, sendo que, com o HNB, não há manipulação de produto amplificado pois é adicionado antes da reação, o que evita possíveis contaminações, e também é possível observar a diferença de cores a olho nu, sem o uso de luz UV. Comparando os custos, em reais, para realização da técnica de PCR e LAMP, esta apresentou um custo de 53,92% menor em relação ao PCR, o que confirma ser uma técnica de baixo custo, já que não requer equipamentos sofisticados e nem eletroforese em gel de agarose para análise dos resultados.Desenvolvemos, neste trabalho, uma técnica para detecção de Wolbachia bastante especifica, rápida, sensível e de baixo custo a qual possibilita seu uso em larga escala para monitoramento de diversas espécies de insetos infectadas no campo...

Detecção da bactéria Wolbachia em insetos através da técnica LAMP (amplificação isotérmica mediada por loop)

Wolbachia pipientis é uma bactéria intracelular que infecta cerca de 40% dos artrópodes e alguns nematódeos, causando alterações reprodutivas em seus hospedeiros. Recentemente, duas cepas de Wolbachia (wMelPop e wMel)foram inseridas individualmente em Aedes aegypti (não infectado naturalmente)e os mosquitos adultos contendo a Wolbachia foram menos suscetíveis a diferentes arboviroses (Dengue e Chikungunya) bem como ao Plasmodium sp. Atualmente, mosquitos A. aegypti contendo Wolbachia (wMel) estão sendo liberados a campo em diversos países, pelo Programa Eliminate Dengue, como possível agente de controle biológico de Dengue. Durante o processo de invasão de mosquitos contendo Wolbachia no campo, há a necessidade de realização de coletas e screening semanal de grandes quantidades de mosquitos para detecção de Wolbachia, via PCR quantitativo, técnica ainda bastante onerosa. Diante disto, é imprescindível o desenvolvimento de um método rápido, específico e de baixo custo para detecção de mosquitos infectados com a bactéria, para levantamento e monitoramento da disseminação da Wolbachia em campo. No presente trabalho, desenvolvemos com sucesso um método rápido de detecção através do LAMP (Amplificação isotérmica mediada por loop) que utiliza 3 pares de primers que se ligam em 8 regiões distintas do DNA alvo, o que torna a reação bastante específica. Em nosso caso, desenhamos iniciadores baseando-se na sequência alvo do gene 16S rRNA da bactéria. Para padronização, foram utilizados mosquitos da colônia do Insetário do Laboratório de Malária do CPqRR, infectados e não infectados por Wolbachia, além de insetos de campo (mosquitos e insetos de diferentes ordens) doados por colaboradores. O tempo de incubação estabelecido para a amplificação foi de 90 minutos à 63oC, sendo possível a realização da reação tanto em termociclador, como em banho seco.

Para análise da sensibilidade, foi feita a diluição seriada de plasmídeo contendo a sequência alvo de 109 à 100 cópias, sendo possível a amplificação utilizando apenas 1 cópia do DNA. Para verificar a especificidade, foram utilizadas amostras de diferentes espécies de bactérias e em nenhuma delas ocorreu a amplificação, confirmando que o LAMP é bastante específico. Uma maneira de reduzir os custos foi através da redução da concentração da enzima Bst DNA polimerase por reação e, com apenas 1 unidade, a amplificação ocorreu de maneira eficiente. Para visualização dos resultados, foram utilizados dois corantes, o azul de hidroxinaftol (HNB) e SYBR Green I. Em ambos foi possível diferenciar as amostras positivas das negativas sem a necessidade de géis de agarose, sendo que, com o HNB, não há manipulação de produto amplificado pois é adicionado antes da reação, o que evita possíveis contaminações, e também é possível observar a diferença de cores a olho nu, sem o uso de luz UV. Comparando os custos, em reais, para realização da técnica de PCR e LAMP, esta apresentou um custo de 53,92% menor em relação ao PCR, o que confirma ser uma técnica de baixo custo, já que não requer equipamentos sofisticados e nem eletroforese em gel de agarose para análise dos resultados.Desenvolvemos, neste trabalho, uma técnica para detecção de Wolbachia bastante especifica, rápida, sensível e de baixo custo a qual possibilita seu uso em larga escala para monitoramento de diversas espécies de insetos infectadas no campo.

Antigen sandwich ELISA predicts RT-PCR detection of dengue virus genome in infected culture fluids of Aedes albopictus C6/36 cells.

Antigen detection by sandwich ELISA was evaluated to predict RT-PCR detection of dengue viral genome in infected culture fluid of Aedes albopictus clone C6/36 cells. Serum specimens collected from dengue patients within 5 days from onset of fever in 2 hospitals in Metro Manila, Philippines, were inoculated into C6/36 cells, and incubated at 28 degrees C. A total of 282 infected culture fluid specimens were harvested and examined by sandwich ELISA and RT-PCR to detect dengue viral antigen and genome, respectively. In the sandwich ELISA, the P/N ratio was calculated by dividing optical density (OD) of a given test specimen by the OD of the standard negative specimen. Samples with a P/N ratio > or = 4.001 were positive for viral genome detection by RT-PCR. The sensitivity and specificity of antigen sandwich ELISA with RT-PCR as the standard, were 90.4% and 100%, respectively. Although antigen sandwich ELISA is less sensitive than RT-PCR, its usefulness lies in its capability to screen a large number of samples at a minimum cost, especially during an outbreak. Samples that meet a set cutoff value can undergo confirmation by RT-PCR for further epidemiological studies.

Morfología y citoquímica de cultivos celulares de Aedes aegypti (Diptera: Culicidae) y susceptibilidad a Leishmania panamensis (Kinetoplastida: Trypanosomatidae)

Morphology and cytochemistry of Aedes aegypti’s cell cultures (Diptera: Culicidae) and susceptibility to Leishmania panamensis (Kinetoplastida: Trypanosomatidae). The first cellular line of Aedes aegypti was developed by Grace in 1966; afterwards, other cellular lines of this species have been generated. These have been used for the study of pathogenic organisms like viruses, bacteria and parasites, which demonstrates their importance in biomedical applications. This research describes, for the first time, some cytochemical characteristics of A. aegypti cell cultures, that were infected with (MHOM/CO/87CL412) strain of Leishmania panamensis. A morphological study of the cell culture was also carried out. Maintenance of the cell culture, parasites and infection in vitro were carried out in the Laboratory of Entomology, Cell Biology and Genetics of the Universidad de La Salle. The cell cultures infected with the parasite were maintained in a mixture of mediums Grace/L15, supplemented with 10 % fetal bovine serum (FBS) at pH 6.8 and a temperature of 26 ºC, during 3, 6 and 9 post-infection days. After this, these cell cultures were processed through High Resolution Light Microscopy (HRLM) and Transmission Electron Microscopy (TEM) based on standard protocols defined by the Group of Microscopy and Image Analyses of the Instituto Nacional de Salud. Semi-fine slices of 1 µm colored with toluidine blue were used for the morphological analysis of the culture, and ultra fine cuts of 60 to 90 nm stained with uranyl acetate and lead citrate where used for the ultrastructural study. In addition, PAS and peroxidase staining was carried out in cells fixed with methanol. The morphometric study was analyzed with software ImageJ (NIH). In the semi-fine slices, small cells were observed showing fibroblastic appearance 10.84±2.54 µm in length and 5.31±1.26 µm wide; other cells had epithelial appearance with a great peripheral nucleus, voluminous and vacuolated cytoplasm, 23.04±4,00 µm in length and 13.96±3.70 µm wide. These last ones predominated over the ones with fibroblastic appearance. Regarding the PAS coloration, 7.08 % of the cells presented abundant PAS positive cytoplasmatic granules which indicated polysaccharides presence. The peroxidase test gave a negative result. The greatest percentage of infection (18.90 %) of one total of 101 cells, turned up by day 6. Some cells analyzed by TEM presented a vacuolated aspect cytoplasm; some contained parasites, other fibrillar material and others were empty. The results indicate that A. aegypti cell culture can support the internalization and transformation of the parasite, which demonstrates the capacity that these cell cultures have to be infected with L. panamensis and to maintain the infection for approximately one week. Rev. Biol. Trop. 56 (2): 447-458. Epub 2008 June 30.

La primera línea celular de Aedes aegypti fue establecida por Grace en 1966 y desde entonces se han utilizado para el estudio de virus, bacterias y parásitos. En el presente trabajo se describen, por primera vez, algunas características citoquímicas de los cultivos celulares de A. aegypti, infectados con la cepa (MHOM/CO/87CL412) de Leishmania panamensis. También se realizó un estudio morfológico de las células del cultivo. Se observaron 30 células pequeñas con apariencia fibrolastoide de 10.84±2.54 µm de largo y 5.31±1.26 µm de ancho; otras 30 presentaron apariencia epitelioide con 23.04±4.00 µm de largo y 13.96±3.70 µm de ancho; éstas últimas predominaron sobre las de apariencia fibroblastoide. De 113 células, un 7.08%, presentaron abundantes gránulos citoplasmáticos positivos con la coloración de PAS, indicando presencia de polisacáridos. La prueba de peroxidasa dio un resultado negativo. El mayor porcentaje de infección (18.90%), de un total de 101 células, se presentó el día 6. Ultraestructuralmente, las células presentaron un citoplasma con aspecto vacuolado; algunas contenían parásitos, otras material fibrilar y otras estaban vacías. Los resultados indican que los cultivos celulares de A. aegypti pueden ser infectados por L. panamensis y mantener dicho proceso por aproximadamente una semana.

Distribución y talla del adulto de Aedes aegypti asociado con los sitios de cría / Distribution and size of adult Aedes aegypti associated with the breeding sites

Introducción: Hoy día se realizan estudios ecológicos con el fin de buscar nuevas estrategias para el control de Aedes aegypti. Objetivos: agrupar los sitios de cría de Ae. aegypti en categorías y determinar el factor de riesgo de estas, establecer la talla de las hembras provenientes de pupas colectadas en el terreno, y conocer sobre la distribución del vector. Métodos: el trabajo se desarrolló en el municipio Playa. Se realizó la inspección de los locales y recipientes de donde se extrajeron las pupas para determinar la talla de las hembras emergidas. Resultados: se clasificaron los depósitos en 5 categorías: depósitos de almacenamiento de agua, depósitos artificiales abandonados en los patios, bebederos de animales, criaderos naturales y gomas. Se revisaron 2 506 locales, de los cuales 57 (2,27 por ciento) resultaron positivos al mosquito para un índice casa de 2,27, un índice de recipiente de 0,53 e índice de Breteau de 2,79. Los mayores valores promedio de longitud del ala de un total de 332 hembras y los mayores valores de factor de riesgo se encontraron en bebederos de animales y gomas con (3,07 ± 0,104 y 3,31 ± 0,95 mm) para un promedio de 2,95 ± 0,25 mm y 2,15 y 5,31, respectivamente. No se encontró diferencia significativa (ANOVA test p= 0,09) al compararse los valores de longitud del ala procedentes de las diferentes categorías de depósitos. Conclusiones: las hembras de Aedes aegypti mostraron variabilidad en su talla. Se encontró este vector distribuido en toda el área estudiada.

Introduction: At present, environmental studies are conducted to find new strategies for the control of Aedes aegypti. Objectives: To classify Aedes aegypti breeding sites into various categories and to determine their risk factors; to determine the size of females from pupas collected in situ and to learn about the vector distribution. Methods: this research work was carried out in Playa municipality. All the premises and water containers were inspected from where the pupas were taken to determine the size of emerged females. Results: Containers were classified into 5 categories: water storage containers, artificial containers left in house yards, troughs for animals, natural breeding sites and tyres. Two thousand five hundred and six premises were visited and inspected and 57 of them (2.27 percent) were positive to the mosquito for a index per house of 2.27; a container index of 0.53 and Breteau´s index of 2.79. The highest average measures in wing length of 332 females and the highest risk factor values were found in troughs for animals and tyres (3.07 ± 0.104 y 3.31 ± 0.95 mm) for an average of 2.95 ± 0.25 mm, and 2.15 and 5.31 respectively. No significant difference was found (ANOVA test, p= 0.09) when comparing the female wing length measurements gathered from the different container categories. Conclusions: Aedes aegypti females showed variable sizes. This vector was spread all over the studied area.

Inhibition of Mayaro virus replication by prostagandin A1 in Aedes albopictus cells

Prostaglandin Al (PGA1) inhibits Mayaro virus replication in Aedes albopictus cells at nontoxic doses to uninfected cells . At 10 ug/ml, PGA1 decreases virus production by 90 (per cent). The presence of PGA1 during virus adsorption, with no treatment after infection, reduces virus yield by 41 (per cent). Antiviral activity is observed even when treatment starts at one or two hours post-infection. However, in cells pre-treated with PGA1 during 24 hours, virus replication is not impaired. Thus, events ocurring during initial stages of infection and after virus adsorption and penetration must be the target of PGA1 action. SDS-PAGE analysis of 35S-methionine labelled proteins shows that PGA1 inhibits the synthesis of viral proteins and induces the synthesis of polypeptides with molecular weight of 70 kDa, 57 kDa and 23 kDa. In cells pre-treated with actinomycin D the induction of those proteins is suppressed. In addition, actinomycin D treatment prevents PGA1 antiviral activity, indicating that PGA1-induced stress proteins are probably involved in this mechanism

Effect of prostaglandin A1 in the induction of stress proteins in Aedes albopictus cells

Prostaglandin are natural fatty acid derivatives with diverse physiological effects, including immune function and the control of cell growth. While the action of prostaglandins in the induction of stress proteins in vertebrate cells in well documented, their functions in invertebrate cells have been poorly investigated. The purpose of the present study was to investigate the effect of prostaglandin A1 (PGA1; 0.25, 1.25 and 12.5 mug/ml) on protein synthesis during the growth of Aedes albopictus cells. We found that PGA1 stimulates the synthesis of several polypeptides with molecular masses of 87,80,70,57,29,27 and 23 kDa in Aedes albopictus cells. When the protein induced by PGA1 and those induced by heat treatment were compared by polyacrylamide gel electrophoresis, PGA1 was found to induce the stress proteins. The HSP70 family and the low-molecular weight polypeptides (29 and 27 kDa, respectively) were induced by PGA1 in the lag phase. We lso observed that PGA1 is able to induce a 23-kDa polypeptide independently of the growth phase of the cell.

Comparison of sequences of E/NS1 gene junction of dengue type 3 virus following culture subpassage in C6/36 cells to study the possible occurrence of mutations.

The aim of this study was to determine whether mutations could occur in the dengue virus genome following three subpassages of the virus in a mosquito cell line. This was done because sources of virus isolates used for sequencing studies are usually maintained in cell lines rather than in patients' sera. Therefore it must be assured that no mutation occurred during the passaging. For this purpose, sequencing was carried out using the polymerase chain reaction (PCR) products of the envelope/non-structural protein 1 junction region (280 nucleotides) of dengue type 3 virus. Sequence data were compared between the virus from a patient's serum against the virus subpassaged three times in the C6/36 cell line. We found that the sequence data of the virus from serum was identical to the virus that was subpassaged three times in C6/36 cell line.

Evaluación de tres técnicas citogenéticas diferentes en los estudios morfométricos del cariotipo de Aedes taeniorhynchus (Diptera: culicidae) / Three different cytogenetic technique evaluation in the morphometrics studies of Aedes taeniorhynchus (Diptera: Culicidae) karyotype

Se efectuaron estudios morfométricos del cariotipo de Aedes taeniorhynchus, mosquito de interés médico-veterinario, por ser vector del virus de la encefalitis equina venezolana, tipo epidemo-epizoótico. En las preparaciones cromosómicas fueron utilizadas tres técnicas citogenéticas diferentes: squash, secado al aire y cultivos celulares. Estas se compararon entre sí para evaluar, en las metafase obtenidas, la longitud y morfología de los cromosomas. El número diploide de la especie fue de seis en todas la preparaciones cariológicas al utilizar cualquiera de los tres procedimientos: sin embargo, con la técnica de squash, los cromosomas mitóticos de cerebro fueron más cortos, discontinuos y con menor grado aparente de compactación de la cromatina; al utilizar la técnica de secado al aire, se obtuvieron mejores extendidos citológicos, cromosomas más separados y ligeramente de mayor longitud; en tanto que los resultados de mejor resolución, por la estructura, separación de homólogos y longitudes cromosómicas, se lograron con cultivos celulares

A rapid membrane based immunobinding assay for the detection of dengue virus in tissue culture.

A rapid, simple dot immunoassay (DOTIA) was developed and evaluated for the detection of dengue-1 viral antigen in infected Aedes albopictus C6/36 cells. Dengue virus infected cells were solubilize in sodium dodecyl sulfate (SDS) and the lysate was pressure filtered through a hydrophobic polyvinylidene difluoride (PVDF) membrane. Viral antigen retained in the membrane was detected by a dengue-1 type specific monoclonal antibody and a peroxidase-labeled second antibody. Addition of tetramethylbenzidene (TMB) substrate produced a blue-colored precipitate which allowed for quantitation of viral antigen using a portable white light reflectance densitometer. Estimate of viral infectivity in the cell lysates tested by the DOTIA was determined by standard plaque assays and the results indicated an excellent correlation between these two methods. The dot immunoassay detected dengue viral antigen in infected C6/36 cells between days three and eight post-inoculation, depending on the titer of the inoculum. An infectivity titer of at least 10(3) plaque forming units (PFU) per ml was required to detect antigen by the DOTIA. The DOTIA also detected viral antigen in cells inoculated with twelve acute sera from known dengue-1 virus infected patients, thus demonstrating that this technique is useful for the detection and identification of dengue-1 virus from clinical specimens.

Translocation of heat shock proteins into isolated nuclei of Aedes albopictus cells

Labelled heat shock proteins of 82 kDa,70 and a group of low molecular weight are present in the post-mitochondrial supernatant obtained from Aedes albopictus cells.These proteins in the post-mitochondrial extract are transported specifically to the nuclear compartment when incubated with isolated unlabelled nuclei.The relative amounts of each protein transported were 4.7,24.1 and 3,1% for the 82-k-Daand 70-kDa and for the low molecular weght proteins, respectively.Incubation of radiolabelled post-mitochondrial extract with unlabelled nuclei at 30 C or 37 C did not modify the perecentage of proteins translocated

Uso de células de Aedes albopictus C6/36 na propagaçäo e classificaçäo de arbovirus das famílias Togaviridae, Flaviviridae, Bunyaviridae e Rhabdoviridae / The use of Aedes albopictus C6/36 cells in the propagation and classification of arbovirus of the Togaviridae, Flaviviridae, Bunyaviridae and Rhabdoviridae families

Colônias de células de mosquito Aedes albopicus C6/36 foram infectadas com 23 arbovirus, sendo 19 destes existentes no Brasil, pertencentes às famílias Togavitidae, Flaviviridae, Bunyaviridae e Rhabdoviridae. A Replicaçäo viral foi detectada por imunofluorescência indireta com todos os vírus estudados enquanto que o efeito citopático foi observado durante a infecçäo por alguns deste. No teste de imunofluorescência indireta utilizou-se fluidos ascíticos imunes de camundongos, especificos para os vírus estudados. A replicaçäo viral caracterizada por grande produçäo de antígeno recomenda a utilizaçäo de células C6/36 na propagaçäo e em tentativas de isolamento desses arbovírus. A técnica de imunofluorescência ofereceu subsídios na classificaçäo e identificaçäo de vírus que replicam nestas células

Development of the second generation Japanese encephalitis (JE) vaccine.

Recent analysis of Japanese encephalitis (JE) virus genome RNA, especially its nucleotide sequence, revealed the localization of virion envelope glycoprotein (E) gene on the virus genome. Since the E protein is the major protective antigen related with the virus neutralization, attempts have been made to produce the second generation JE vaccine by expressing the E protein using recombinant DNA technologies. These studies will eventually lead to control JE by mass-vaccination in several Asian countries where JE is one of the major public health problems.